Fig 2: Tumor-infiltrating immune cells and their role in tumor growth. Analysis of tumor-infiltrating cells was performed by flow cytometry (A,B). Mice (n = 4) were injected with tumor cells and immunized by a gene gun. Vaccine adjuvants and anti-Tim-3 were administered on the same days as the DNA vaccines. Tumor cells were isolated on day 12 from non-treated tumors and on days 14–18 from treated tumors and stained with fluorochrome-labeled antibodies. (A) Frequencies of CD45+ and CD3+ cells, Treg (CD4+CD25+Foxp3+) and Nrp1+ Treg cells. Statistical significance refers to the comparison with the non-treated (pBSC) group. (B) Overview of the mean percentages of the major subpopulations of tumor-infiltrating cells in total cells. (C) The effect of in vivo depletion of immune cells and neutralization of IFN-? on the anti-tumor response induced by immunotherapies with ODN1826 or a-GalCer in the immunized mice (n = 5). Vaccine adjuvants were injected on the days of immunization. Statistical significance refers to the comparison with the group that was immunized with the PADRE.E7GGG gene and received an adjuvant. Bars: ±SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3: Loaded NPs promote self-specific CD8+ T cells expansion and activation in vitro and reduce systemic toxicities of a soluble stimulant mixture in vivo. (A) The TLR1/2 agonist and NOD2 agonist stimulated self-antigen–specific CD8+ T cells in healthy human PBMCs (n = 5). In this assay, PBMCs were directly cultured with Pam3CSK4 and L18-MDP in the presence of pooled self-specific peptides, anti-CD28 Ab, and IL-2 and stained with pooled HLA-A*0201 tetramers at day 7. The T cell activation was characterized by measuring the percentage increase in CD25 expression of self-tetramer+CD8+ T cells. Data are means ± SD. Data were analyzed by unpaired t test with Welch's correction. **P < 0.01. (B) The representative flow cytometric analysis showing the proliferation and activation of self-specific CD8+ T cells from healthy blood donors after stimulation with a mixture of soluble stimulants (Top) or NPs (Bottom) in the presence of pooled self-peptides at day 7.5. The soluble stimulant mixture includes IL-2, Pam3CSK4, L18-MDP, and anti-CD28 Ab. The NPs were loaded with equivalent dose of IL-2, Pam3CSK4, and L18-MDP, and anti-CD28 Abs conjugated to their surfaces. For the proliferation assay, self-specific CD8+ T cells were sorted by pooled HLA-A*0201 tetramers loaded with self-specific peptides, CFSE labeled, and cultured under the different stimulation conditions in the presence of autologous feeder PBMCs with the same peptides used for tetramer sorting. T cell proliferation was characterized by measuring the CFSE dilution of tetramer+CD8+ T cells. The most brightly CFSE-stained cells are not proliferating. For the activation assay, PBMCs were directly cultured under the stimulation conditions in the presence of pooled self-specific peptides and stained with pooled HLA-A*0201 tetramers at day 7.5. The T cell activation was characterized by measuring the percentage increase in CD25 expression of self-tetramer+CD8+ T cells. Data are representative of three human individuals. (C) Representative flow cytometric analysis showing the proliferation of TRP2-specific CD8+ T cells isolated from B16F10 melanoma-bearing mice. CD8+ T cells were sorted from tumor-draining lymph nodes of B16F10 melanoma-bearing mice, labeled with cell trace far red (CTFR), and stimulated with different stimulants such as anti-CD28 Ab plus TRP2 peptide, anti-CD3/CD28 Ab, or NPs plus TRP2 peptide. Data are representative of two independent experiments. (D) The concentration of IL-6 in the serum of C57BL/6 mice 2 h after a single subcutaneous injection of a soluble stimulant mixture or NPs (n = 3). The data were represented as fold change of mean fluorescence intensity (MFI) over the PBS group. (E) Splenomegaly assessment of C57BL/6 mice on day 7 received three injections (day 1, day 3, and day 5) of PBS, NP, or a soluble stimulant mixture. Soluble is short for a soluble stimulant mixture (n = 3). Data are means ± SEM. Data were analyzed by one-way ANOVA with Bonferroni posttest. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Differential Regulation of IL-17 and IL-22 in CD18-/- Colon after C. rodentium Infection(A and B) Eight-week-old CD18-/- mice and CD18-sufficient littermate controls (CD18+/- and/or CD18+/+) were orally inoculated with C. rodentium. On day 5, colonic tissue was harvested, and expression of the indicated cytokines was determined (A) at the mRNA level by quantitative real-time PCR and (B) at the protein level by ex vivo colon culture ELISA.(C and D) On day 5 post-infection, LPMCs were isolated from the large intestine of CD18-/- or CD18+/- mice and analyzed by flow cytometry for IL-22+ ILC3 cells (shown are representative fluorescence-activated cell sorting [FACS] plots [left] and absolute numbers [right] of IL-22-expressing ROR?t+ ILCs [gated on CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD127+CD90.2+ROR?t+]) (C) and for total number of ILC3 cells (D).Data are means ± SDs and are pooled from 2 independent experiments with 3 mice/group in each experiment, for a total of 6 mice/group (A and B); are from 2 independent experiments with 3 mice/group in one experiment and 4 mice/group in the second experiment, for a total of 7 mice/group (C); or are from 3 independent experiments with 3 mice/group in each experiment, for a total of 9 mice/group (D). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (A, C, and D: 2-tailed Student’s t test; B: or 1-way ANOVA with Tukey’s multiple-comparisons test). NS, non-significant.

Fig 5: Single-Cell RNA Sequencing of Colonic Macrophages, Related to Figure 3(A, left) tSNE computed on the top 22 PCs obtained on the 1000 most variable genes (vst method). Cells are colored by the sample. (A, right) tSNE computed on the top 22 PCs obtained on the 1000 most variable genes (vst method). Cells are colored by the cluster (number of neighbors = 30, resolution = 0.3).(B) Distribution of the log-normalized expression levels of genes used for sorting terminally-differentiated ?? across clusters (gated on alive, CD45+, CD3-, CD19-, CD103-, CD11b+, F4/80+, CD64+, Ly6C-, MHCII+).(C, top panel) Distribution of the log-normalized expression levels of fibroblast/myofibroblasts markers across clusters. (C, bottom panel). Distribution of the log-normalized expression level of epithelial cell markers across clusters.(D) Clusters 4 and 6 from Kang et al., consist of terminally differentiated (mature) M?. Venn diagram of the set of cluster 4, 6 and 7 markers (top 50 upregulated genes, gene AF251705 is excluded because it is not associated to any official gene name, Kang et al.) and the terminally differentiated macrophage signature, defined as the genes showing log2(FC) 3 3 from P1 (monocytes) to P4 (mature ??) by Schridde et al. (65 genes).